Table 1.
Percentage of each cell type used to form cardiac spheroids in this study.
Fig 1.
Cells aggregate as spheroids (A). The desired 3D design is composed using the needle array software (B) and the appropriate needle array (C) is selected and prepared. The spheroids are then printed onto the needle array (D).
Fig 2.
Motion analysis of the contractile area in cardiac spheroids and constructs.
The magnitude of each contraction in the cardiac spheroids and constructs was recorded and individual frames were isolated (A). The spheroid’s and construct’s area were then measured in each frame using our in-house software (B). The fractional area changes in the spheroids and constructs were calculated and graphed (C).
Fig 3.
Electrical stimulation system.
Basic diagrams of the electrical stimulation system (A) and the electric circuit (B). The electrical stimulator generates pulses which are then transmitted to the medium in the dish. To image the constructs, the samples were placed between the Pt electrodes and the entire system was set on a microscope (C).
Fig 4.
Time-lapse imaging of cardiac spheroid formation.
Cardiac spheroids containing different percentages of iCells, HUVECs, and NHDFs were prepared and their formation process was imaged and recorded on days 1, 3, 5, and 7.
Fig 5.
Spheroid size and roundness change over time.
Changes in size (A) and roundness (B) were measured in the cardiac spheroids composed of the specified cell mixtures for 9 days. The size of all cardiac spheroids was measured at day 7, before being used to fabricate the cardiac constructs (C).
Fig 6.
Motion analysis of the contractile cardiac spheroids.
The contraction in five types of spheroids were recorded (A), and the beat rate (B) and contraction (C) were analyzed.
Fig 7.
Histological and immunohistochemical analysis of five types of cardiac spheroids.
Cardiac spheroids containing different percentages of iCells, HUVECs, and NHDFs were fixed and stained after 7 days. Troponin T is a marker for iCells, while CD31 and CD90 are markers for HUVECs and NHDFs, respectively.
Fig 8.
Culture of tubular cardiac constructs on a needle array.
Representative images of the fabricated tubular cardiac constructs just after printing (A, B) and after being cultured on the needle array for 7 days (C, D).
Fig 9.
Culture and maturation of tubular cardiac constructs on the plastic catheters.
Representative images of the tubular constructs immediately (A), 1 day (B), and 6 days (C) after removal from the needle array as well as after removal from the plastic catheters (D). The areas of the tubular construct were examined (E).
Fig 10.
Analysis of the changes in beat rate in response to electrical stimulation of the tubular cardiac constructs.
The tubular cardiac constructs were removed from the needle array (A) and cultured for 7 days (B). Electrical stimulation was then applied and its effects were measured and analyzed (C).
Fig 11.
Immunohistochemical analysis of the cardiac constructs.
Representative images of spheroids and tubular constructs fabricated with iCells, HUVECs, and NHDFs for 21 days are shown. These samples were observed at low (4×) and high (20×) magnification. iCells were identified with troponin T staining, while HUVECs and NHDFs were stained with CD31 and CD90, respectively.